The invention relates to 169 genes identified by their co-expression with genes known to be involved in bone remodeling and bone diseases. The invention also relates to the use of these genes and their gene products in diagnosis, prognosis, prevention, treatment, and evaluation of therapies for bone diseases such as osteoporosis.
Bone Remodeling
Bone remodeling occurs through teams of juxtaposed bone absorbing osteoclast and bone forming osteoblast and osteocyte cells. The development and proliferation of these cells from their progenitors is governed by networks of growth factors and cytokines produced in the bone microenvironment as well as by systematic hormones (Manolagas (1998) Aging 10:182-190; Teitelbaum et al. (1997) J Leukoc Biol 61:381-388). Coordinated balance between absorption and deposition is necessary to maintain bone integrity and requires intimate and complex interactions between osteoclasts and osteoblasts. Under normal states of bone homeostasis, the remodeling activities in bone serve to remove bone mass where the mechanical demands of the skeleton are low and form bone at the those sites where mechanical loads are repeatedly transmitted.
Bone is a composite material composed of an organic and an inorganic phase. By weight, approximately 70% of the tissue is mineral or inorganic matter (mainly calcium phosphate); water comprises 5 to 8%; and, the organic or extracellular matrix makes up the remainder. Approximately 95% of the mineral phase is composed of a specific crystalline hydroxyapatite that is impregnated with impurities which make up the remaining 5% of the inorganic phase. Ninety-eight percent of the organic phase is composed of type I collagen and a variety of non-collagenous proteins; cells make up the remaining 2% of this phase (Einhom (1996) The bone organ system: form and function. In: Marcus et al. eds., Osteoporosis, Academic Press, New York N.Y.). The process of matrix deposition by osteoblasts and osteocytes, subsequent mineralization and the coupling with bone resorbing activity of osteoclasts is governed by a complex interplay of systemic hormones, peptides and downstream signaling pathway proteins, local transcription factors, cytokines, growth factors and matrix remodeling genes.
Parathyroid hormone (PTH) and it""s signaling system is the principal regulator of bone remodeling in the adult skeleton (Masiukiewicz and Insogna (1998) Aging 10:232-239; Mierke and Pellegrini (1999) Curr Pharm Des 5:21-36). It has a vital role in the homeostasis of calcium within the blood stream and acute in vivo effect of PTH is to increase bone resorption, although sustained increases in its circulating levels accelerate both formation and resorption. The PTH signaling pathway may also be involved in the regulation of chondrogenesis during bone formation (Vortkamp et al. (1996) Science 273:613-622; Lanske et al. (1999) J Clin Invest 104:399-407).
Several other hormones and local factors are vital to bone health. In a complex pattern of inhibition and stimulation not yet fully understood, growth hormone, insulin-like growth factor-1, the sex steroids, thyroid hormone, calciotrophic hormones such as PTH and prostaglandin E2, various cytokines, such as interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha, and 1,25-dihydroxyvitamin D (calcitriol) all act coordinately in the bone remodeling process. Estrogen is involved in inhibition of osteoclast activity (Jilka et al. (1992) Science 257:88-91; Poli et al. (1994) EMBO J 13:1189-1196; Srivastava et al. (1998) J Clin Invest 102:1850-1859). Estrogen may prevent bone loss by blocking the production of cytokines in bone or bone marrow (Kimble et al. (1995) Endocrinology 136:3054-3061). Various cytokines, such as interleukin-l beta, interleukin-6, and tumor necrosis factor-alpha, influence bone remodeling (de Vemejoul (1996) Eur J Clin Chem Clin Biochem 34:729-734).
Bone Remodeling and Osteoporosis
Throughout life, old bone is removed (resorption) and new bone is added to the skeleton (formation). During childhood and teenage years, new bone is added faster than old bone is removed. As a result, bones become larger, heavier and denser. Bone formation continues at a pace faster than resorption until peak bone mass is reached during the mid-20s. After age 30, bone resorption slowly begins to exceed bone formation, most rapidly in the first few years after menopause but persistantly until death. Osteoporosis develops when bone resorption occurs too quickly or if replacement occurs too slowly. Two major classes of osteoporosis are primary and secondary osteoporosis. Type I osteoporosis occurs in a subset of postmenopausal women who are between 50 and 70 years of age and is associated with fractures of vertebral bodies and the forearm. Type II osteoporosis occurs in women and men over the age of 70 and is associated with fractures of the femoral neck and proximal humerus and tibia. In some instances, osteoporosis is a manifestation of another disease (Fauci et al. (1998) Harrison""s Principles of Internal Medicine, McGraw Hill Companies, New York N.Y., pp 2249). Current therapies are designed to interfere with these growth regulatory systems to encourage the growth and function of osteoblasts and inhibit the growth and activity of osteoclasts.
The present invention satisfies a need in the art by providing new compositions that are useful for diagnosis, prognosis, treatment, prevention, and evaluation of therapies for bone remodeling and associated disorders, such as osteoporosis. We have implemented a method for analyzing gene expression patterns and have identified 169 polynucleotides by their co-expression with genes known to be involved in bone remodeling and osteoporosis.
The invention provides for a combination of substantially purified polynucleotides that are co-expressed with one or more genes known to be involved in bone remodeling and osteoporosis wherein the polynucleotides or the complements thereof are SEQ ID NOs: 1-169. The genes known to be involved in these conditions include genes encoding PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), human parathyroid hormone, TNF receptor associated factor 2, tumor necrosis factor receptor superfamily member 11/RANK ligand, tumor necrosis factor receptor superfamily member 11A/receptor activator of NFkB (RANK), vitamin D 24-hydroxylase, natriuretic precursor protein B. human granulocyte macrophage colony stimulating factor 2, cytochrome P450, subfamily XIX, epidermal growth factor, interleukin 1 alpha, microphthalmia-associated transcription factor, bone morphogenic protein 7, human integrin-binding sialoprotein beta-3 integrin, alpha-2-Hs-glycoprotein, osteoprotegerin, interleukin 1 beta, SPI1, matrix metalloprotease 9, parathyroid receptor 1, SRC kinase insert domain receptor, vascular endothelial growth factor receptor, disintegrin and metalloprotease 12, human homolog of Drosophila mothers against decapentaplegic 6, parathyroid hormone receptor 2, glucocorticoid receptor-like 1, phosphodiesterase I, bone morphogenic protein 2, vitamin D receptor, human estrogen receptor 1, TNF receptor associated factor 5,transforming growth factor beta 1, acid phosphatase type 5, cathepsin K, bone morphogenic protein 6, secreted protein, acidic, cysteine-rich, osteonectin, vascular endothelial growth factor A, vascular endothelial growth factor receptor, bone gla protein (osteocalcin), carbonic anhydrase II, alpha-V integrin, early growth response 1, collagen type 1 alpha 2, macrophage colony stimulating factor 1, biglycan, secreted phosphoprotein (osteopontin), insulin growth factor-1, decorin, cathepsin B, colony stimulating factor receptor, and Klotho. The invention also provides a combination wherein the polynucleotides or the complements thereof are SEQ ID NOs: 107-169 that are highly co-expressed with genes known to be involved in bone remodeling and osteoporosis. The invention further provides a combination wherein the polynucleotides or the complements thereof are SEQ ID NOs: 143-169 that are very highly co-expressed with genes known to be involved in bone remodeling and osteoporosis. The invention additionally provides novel polynucleotides or complements thereof selected from SEQ ID NOs: 155-169 which are co-expressed with one or more of the genes known to be involved in bone remodeling and osteoporosis.
The invention provides a method of using a polynucleotide selected from SEQ ID NOs: 155-169 to screen a library of molecules or compounds (DNA molecules, RNA molecules, PNAs, mimetics, and proteins) to identify or purify a ligand which specifically binds the polynucleotide by combining a polynucleotide with a library of molecules or compounds under conditions to allow specific binding, and detecting specific binding, thereby identifying or purifying a ligand which specifically binds the polynuclcotide.
The invention provides for the use of at least one polynucleotide selected from SEQ ID NOs: 1-169 on an array and for the use of that array in a method for diagnosing a bone remodeling disease or disorder by hybridizing the array with a patient sample under conditions to allow complex formation, detecting complex formation, and comparing the amount of complex formation in the patient sample to that of standards for normal and diseased tissues wherein the complex formation in the patient sample indicates the presence of a bone remodeling disease or disorder.
The invention also provides for an expression vector comprising a polynucleotide selected from SEQ ID NOs: 1-169, a host cell transformed with the expression vector and a method for producing a polypeptide, the method comprising: culturing the host cell under conditions for expression of the polypeptide and recovering the polypeptide from cell culture.
The invention further provides for a polypeptide selected from SEQ ID NOs: 170-172, or a portion thereof, comprising the product of a gene that is co-expressed with one or more genes known to be involved in bone remodeling and osteoporosis. The invention additionally provides for the use of the polypeptide or a portion thereof to screen a library of molecules or compounds (DNA molecules, RNA molecules, PNAs, mimetics. proteins, agonists, antagonists, and antibodies) to identify or purify at least one ligand which specifically binds the polypeptide by combining the polypeptide or a portion thereof with the library of molecules or compounds under conditions to allow specific binding, and detecting specific binding between the polypeptide and ligand, thereby identifying or purifying a ligand which specifically binds the polypeptide.
The invention finally provides for a pharmaceutical composition comprising a polynucleotide selected from SEQ ID NOs: 1-169, polypeptide selected from SEQ ID NOs: 170-172, a ligand identified or purified using a selected polynucleotide or polypeptide which modulates the activity of the selected polynucleotide or polypeptide and a suitable pharmaceutical carrier.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The Sequence Listing provides exemplary polynucleotide sequences, SEQ ID NOs: 1-169, and polypeptide sequences, SEQ ID NOs: 170-172, which co-express with genes known to be involved in bone remodeling and osteoporosis. Each sequence is identified by a sequence identification number (SEQ ID NO) and Incyte ID No.